key: cord-0719565-qylpzgfu authors: Klasse, Per Johan; Bron, Romke; Marsh, Mark title: Mechanisms of enveloped virus entry into animal cells date: 1998-10-05 journal: Adv Drug Deliv Rev DOI: 10.1016/s0169-409x(98)00002-7 sha: d3aaeac6529047c54bc3c641043415d8f83e175c doc_id: 719565 cord_uid: qylpzgfu The ability of viruses to transfer macromolecules between cells makes them attractive starting points for the design of biological delivery vehicles. Virus-based vectors and sub-viral systems are already finding biotechnological and medical applications for gene, peptide, vaccine and drug delivery. Progress has been made in understanding the cellular and molecular mechanisms underlying virus entry, particularly in identifying virus receptors. However, receptor binding is only a first step and we now have to understand how these molecules facilitate entry, how enveloped viruses fuse with cells or non-enveloped viruses penetrate the cell membrane, and what happens following penetration. Only through these detailed analyses will the full potential of viruses as vectors and delivery vehicles be realised. Here we discuss aspects of the entry mechanisms for several well-characterised viral systems. We do not attempt to provide a fully comprehensive review of virus entry but focus primarily on enveloped viruses. cess of entry of the vehicle into cells. Thus a more detailed understanding of virus entry may suggest Currently, viruses are used as delivery vehicles in ways in which existing delivery systems can be two different ways. Firstly, viral genomes have been improved or novel ones developed. manipulated so that foreign genes can be inserted and the modified genome packaged to generate virus particles that resemble the original viruses. These 2. Strategies for virus entry virions then follow the pathways used by the parental virus to infect a target cell. Such systems are well Virus entry involves binding of virions to the established for retroviral vectors, and for a variety of surface of an appropriate cell and delivery of the other virus systems including adeno-, adeno-associ-viral nucleic acid to the cytoplasm or nucleus of that ated virus, alpha-, vaccinia, and herpes virus. As an cell. Frequently viruses are taken up by endocytosis. alternative strategy, components deemed to be re-However, virions within membrane-bound endocytic quired for viral entry have been isolated and used to vesicles remain topologically outside the cytoplasm facilitate delivery. For example, the influenza en-until the events that lead to penetration are triggered. velope glycoprotein, haemagglutinin (HA), has been In this review we will use the term 'penetration' to used to make virosomes (liposomes containing re-describe the process whereby viral nucleic acid is constituted viral membrane proteins that can be transferred across a cellular membrane into the loaded with peptides, drugs, DNA, etc.) that are as cytosol of the target cell. The term 'internalisation' is effective for fusion as the original virions reserved for the endocytosis of virions, a process that [27, 199, 217, 225] . Peptides representing an HA fu-may or may not lead to productive infection but does sion motif have been coupled to DNA and used to not itself constitute viral entry. deliver genes to tissue culture cells [169, 221] . Similarly, inactivated adenovirus-ligand-DNA conju-2.1. Enveloped viruses gates, in which the virus acts as a carrier and not an infectious particle, have been developed [145] . Al-Viruses have evolved many strategies to invade though these systems work well in tissue culture, and cells. In a few cases the cellular and molecular in some cases in vivo, they lack the specificity and details are reasonably well understood, but for most efficiency required for clinical applications. In many viruses little is known. Nevertheless, the well-studied cases these limitations are associated with the pro-systems have established paradigms against which other viruses can be compared. The entry strategies and the target membrane (the so-called vesicle (v)used by different viruses are determined primarily by and target (t)-SNARES) have been implicated as the structure of the virus. Viruses are either en-receptors that ensure transport vesicles fuse with the veloped or non-enveloped. Enveloped viruses con-correct compartment [177] . Similarly, the membrane tain the viral genome and core proteins wrapped of enveloped viruses must also contain the comwithin one or more membranes. These membranes ponents required for (i) binding to, and (ii) fusing are acquired from the host cell during virus assembly with a target cell. and budding [166] . Many enveloped viruses, such as the orthomyxo-(e.g., influenza), paramyxo-(e.g., 2.2. Non-enveloped viruses Sendai), rhabdo-(e.g., vesicular stomatitis virus), retro-(e.g., human immunodeficiency, [HIV] ) and Per definition, non-enveloped viruses do not conalphaviruses [e.g., Semliki Forest virus (SFV)]), tain membranes (though rotaviruses may transiently contain a single membrane. Other viruses, such as acquire a membrane by budding into the ER, only to herpes virus, may undergo several budding and lose the membrane during subsequent maturation fusion steps with different intracellular membrane [211] ). The viral genome is incorporated into a compartments before finally acquiring a single mem-protein shell in the cytoplasm or nucleoplasm of the brane from the exocytic pathway. In contrast, vac-infected cell and the assembled virions are then cinia virus (a member of the Poxviruses) acquires released by cell lysis or, for Rotaviruses, secretion. several membranes by interaction with different Like viral envelopes, the outer protein shell of membrane compartments in the infected cell non-enveloped viruses must contain the necessary [183, 192, 214] . The composition of the viral mem-molecular equipment for getting into cells. However, brane(s) varies for different viruses and is determined unlike enveloped viruses, non-enveloped viruses by (i) the complement of virally encoded membrane must rely on strategies other than fusion. In general, or envelope proteins and (ii) the content of host cell proteins and protein complexes are not spontaneouslipid and proteins. The latter reflects (i) the degree to ly transported across membranes but require complex which cellular proteins are incorporated into the molecular machines for translocation. The mechabudding virion and (ii) the composition of the nisms through which the protein shells of non-enmembrane system where budding occurs [different veloped viruses interact with cell membranes are not viruses assemble and bud in different cellular loca-well understood, and there is no single principle tions, for example the endoplasmic reticulum (ER), analogous to fusion. Picornaviruses bind to cellintermediate compartment, Golgi apparatus and plas-surface components that can dock into 'canyon-like' ma membrane [166] ]. depressions on the surface of the virion [155, 176] . Enveloped viruses use membrane fusion to pene-Following endocytosis, the virions appear to generate trate a cell [239] . The viral membrane fuses with a pores in the endosome membrane that allow the viral cellular membrane so that the genome-containing RNA to exit from the virion and penetrate to the viral capsid or core is transferred to the cytosol. cytoplasm without complete disassembly of the Membrane fusion is one of the most frequent re-capsid or disruption of the endosomal membrane actions occurring in eukaryotic cells. Cell-to-cell [171] . Receptor binding may initiate changes in the fusions, such as sperm-oocyte or myoblast fusion, capsid structure that facilitate pore formation. In are topologically closely related to virus entry [240] . contrast, adenoviruses employ fibres that project However, membrane fusion events occur at higher from the virion surface to bind to cellular receptors. frequency inside cells as vesicles transporting mem-After initial adsorption to the cell surface, adenobrane and cargo between intracellular organelles are viruses bind to vitronectin-binding integrins and formed and consumed [177] . In principle, the cycle undergo endocytosis through coated vesicles. Within of virus assembly and fusion is similar to that of endosomes the low pH activates a lysin on the virus transport vesicle formation and consumption. Re-that disrupts the endosome membrane allowing the cently membrane proteins on the transport vesicle virion and other endosomal contents into the cyto-plasm where the viral capsid is subsequently dis-Epstein-Barr virus appears to use an endocytic assembled [78, 79] . route to infect lymphocytes, but fuses at the surface of epithelial cells [137] . Similarly, a murine ecotropic retrovirus is apparently pH-independent for 2.3. Cellular sites for penetration: Acid-dependent entry on some cell types but pH-dependent on others versus acid-independent penetration [130] . The causes of these differences are unclear. Penetration may depend on conditions other than the Although enveloped and non-enveloped viruses pH of intracellular organelles. For example, endouse different mechanisms to enter cells, penetration somal proteases have been suggested to activate can occur either at the cell surface or from endocytic Moloney murine leukemia virus fusion [4]. Thus organelles (mainly early and late endosomes). Which although pH-dependent viruses only penetrate from of these sites is used varies for different virus endocytic organelles, it should not be assumed that families. The fusion reactions of enveloped viruses pH-independent viruses only penetrate at the cell are mediated by specific virally encoded envelope surface. proteins. These fusion proteins are displayed on the virion in metastable forms that must be activated to initiate fusion. For alpha-, orthomyxo-, and rhab-2.4. Weak bases and other agents that raise the doviruses, at least, fusion is triggered by exposure of pH of acidic organelles the fusion proteins to low pH and thus usually occurs only after virions have undergone endocytosis into For many pH-dependent viruses entry has been acidic endocytic organelles. Fusion is activated in investigated using weak bases, ionophores or specific 1 early (pH . 6.0) or late endosomes (pH , 6.0) de-inhibitors of vacuolar-type H -ATPases (vATPases, pending on the pH requirement of the specific the enzymes responsible for acidification of intracelenvelope protein [102] . For these viruses endocytosis lular organelles) such as Bafilomycin A and Conis essential for infection. canamycin A. Though their modes of action differ, Not all enveloped viruses are pH-dependent for all these agents raise the pH of acidic organelles and entry. Coronaviruses and paramyxoviruses, as well inhibit pH-dependent viral penetration [85, 100] . as most retroviruses, including the primate immuno-They do not directly affect pH-independent viruses deficiency viruses [HIV-1, HIV-2 and the simian [129] , though they may influence infection indirectly. immunodeficiency viruses (SIV)] do not require low Modulation of endosomal pH, for example, can pH to activate fusion. The fusion triggers in these affect vesicular transport between endocytic comcases are unknown but may involve interaction of the partments. The weak base primaquine interferes with viral envelope proteins with specific receptors (see recycling from endosomes [89, 204] , while neutralisabelow). Some pH-independent viruses appear to be tion of endosomes appears to inhibit transport from capable of fusion across a range of pH values early to late endosomes under some circumstances [129, 239] and may potentially fuse at the cell surface [39] . Blockade of this latter step, using specific or within endocytic organelles. The pH optimum for antibodies, can inhibit vesicular stomatitis virus cell-to-cell fusion mediated by the envelope protein infection [242] . Thus perturbation of intracellular (Env) of a T cell line-adapted HIV-1 strain is around transport by agents that neutralise intracellular or-pH 7.5, well above the pH of endosomes [70] , and ganelles may prevent viruses from reaching sites such strains of HIV-1 enter HeLa-CD4 cells by necessary for penetration or infection. The pH-neufusion at the cell surface [156, 165] . However, the tralising agents can also perturb the assembly of pH-dependencies of other strains of this virus are viruses by neutralising exocytic compartments in unknown and, given the observations of HIV in cells (see, for example, Ref. [129] ). Thus entry endocytic organelles in various cell types experiments that use release of infectious virus as a [22, 74, 173] , it remains possible that some may use read-out should be designed so that the agents are the endocytic route for infection. only present during the time of virus entry. capacity of a virus to replicate in specific types of cells, i.e. to enter them and subsequently produce Virus entry is initiated when a virion first binds to infectious progeny virus. Although the use of spethe surface of a potential host cell. The events that cific receptors accounts for viral tropism in many occur during this first encounter are unclear and instances, post entry effects can also be responsible likely to vary for different viruses. Viral attachment (see, for example, Refs. [23, 178] ). is mediated on the one hand by binding proteins exposed on the surface of the virus particle (we refer 3.1. Viral attachment proteins (VAPs) to these as viral attachment proteins-VAPs), and on the other by 'virus receptors' expressed on the target Enveloped viruses attach to cell surface receptors cell (Fig. 1 ). The idea that specific cell surface through one or more of their envelope glycoproteins. components could be used as 'virus receptors' was Many viruses contain just a single species of eninitially proposed to explain viral tropism-the velope glycoprotein complex that must mediate both the receptor binding and fusion functions, e.g. the ever, as yet there is no method to translate these spike glycoprotein complex of alphaviruses, and the binding constants into the functional affinity of trimeric glycoprotein (G protein) of rhabdoviruses. whole virions for receptor-positive cells because of The primate lentiviruses carry one species of virally the potential multivalency of that interaction. encoded envelope protein (Env-gp120 / gp41), but pick up a range of cellular proteins during assembly, 3.2. Virus receptors including MHC class II antigens [9]. Although not essential, these additional proteins may facilitate Table 1 lists the cell surface components for binding to a host cell and may be targets for which a role in enveloped virus entry has been antibodies with apparent antiviral activities [9]. In established. This table indicates that viruses can use the paramyxoviruses, binding and fusion are per-a variety of different surface moieties including formed by different proteins, and more complex glycoproteins, glycolipids and phospholipids, though viruses such as herpes, may carry several different the majority are glycoproteins. Of these, representa-VAPs and fusion proteins [184] . tives of most classes of protein can be used; single To date the best-characterised VAP is influenza pass type 1 integral membrane proteins (e.g., CD4), virus HA. A pocket in the globular head of the HA1 multispanning proteins (e.g., amino acid transporters) subunit binds the terminal sialic-acid residue of and glycophosphatidylinositol (GPI)-linked proteins oligosaccharides attached to cell surface glycolipids [e.g., the low density lipoprotein receptor (LDLR)and glycoproteins. The 3D structure of HA (from the related protein]. Many virus receptors are members X-31 strain of influenza virus), complexed with of the immunoglobulin super family, molecules that sialyllactose, has been resolved by X-ray diffraction. are often implicated in recognition events; others are In this structure sialic acid completely fills the pocket transporters, components of the glycocalyx, adhesion and is co-ordinated by residues that line the cavity molecules, etc. Among the receptors for retroviruses [231] . The receptor interactions with canyon-like alone, molecules such as CD4, multimembrane-spansites on the surfaces of picornaviruses, in particular ning transporters and a glycolipid-anchored homorhinoviruses, have also been structurally determined logue of the LDLR are used by viruses with similar by crystallography and electron microscopy SU-TM complexes. This disparity notwithstanding, [155, 176] . For many other viruses, however, in-one unifying trait among these interactions has been formation on receptor binding is less direct. For suggested: three crucial residues in CD4, the ecoexample, extensive studies of HIV-1 Env using tropic MuLV receptor (cationic amino acid transporsoluble forms of the major receptor for the virus, ter) and the ALV receptor (LDLR-related protein) CD4, and anti-Env monoclonal antibodies have encompass one aromatic residue and at least one identified continuous and discontinuous epitopes that charged residue which may reflect similarities in are likely to be involved in CD4 binding (Ref. [142] well-adapted modes of viral receptor binding and references therein). These studies suggest the [233, 256, 261] . CD4 binding site is assembled from different regions Overall there are no clear patterns in the usage of of the gp120 molecule, some of which are far apart particular classes of cell surface molecules by speon the linear sequence. cific viruses or virus families, and it is perhaps VAP-receptor binding interactions have been ex-surprising that pH-dependent viruses are not replored using intact virus and soluble forms of the stricted to using receptors containing well character-VAPs and of the receptors. Of these the HIV gp120-ised endocytosis motifs. Indeed, poliovirus infection, CD4 interaction is among the most intensely studied. which is pH-dependent, can be mediated by a With a number of different gp120 preparations the poliovirus receptor that lacks the cytoplasmic tail dissociation constant for monomeric gp120(SU) [107] . This suggests that cell surface molecules, binding to CD4 is in the nanomolar range [191] . For regardless of whether they contain an endocytosis a soluble form of the receptor, sCD4, binding to signal, will be internalised when cross-linked by oligomeric SU anchored to TM on a cell [53, 104] or multiple VAPs. virion surface [140] , it is somewhat higher. How-In some cases the viral binding site on a receptor may coincide with the binding site for the physiolog-surface. However, in a number of cases specific ical ligand, e.g. Epstein-Barr virus and complement VAP-receptor interactions are required to facilitate factor C3dg bind to overlapping sites on complement viral entry. Specific receptor interactions may be receptor 2 [144, 147] . Chemokines that bind the co-required to initiate conformational changes, or other receptors for HIV (see below) can inhibit virus entry events, leading to fusion and / or penetration. For [19, 40, 52, 57, 152] , though ligand-induced receptor example, the interaction of poliovirus with a soluble endocytosis may be involved in this process [187] . form of its receptor leads to partial dissociation of The partial inhibition of the physiological function of the viral capsid structure [107] , suggesting that virus receptors by VAP binding has been described docking to the receptor may have some role in for the amino-acid and phosphate transporter mole-unlocking the capsid structure to allow the viral cules that serve as receptors for several retroviruses RNA out. [95, 153, 227] . In addition to the use of cell surface components Virus receptors may just tether virions to the cell in entry, there are examples of VAPs that bind specifically to cellular receptors that do not function 2). The exact nature of these additional interactions in viral entry. For example, the Friend erythroleuke-again varies for different viruses. For HIV, interacmia virus VAP binding to the erythropoietin receptor tions with chemokine receptors post-CD4 binding can activate the receptor, but cannot mediate in-appears to be essential for fusion (see below). For fection [111] . Herpes simplex virus (HSV), heparan-sulphate glycosaminoglycans can function to recruit the virus 3.3. Alternative receptors [253] but a novel tumour necrosis factor / nerve growth factor receptor homologue appears to be In many systems, viruses appear to bind directly to required for entry [139] . Similarly, initial high the cell surface molecules that mediate internalisa-affinity binding of adenovirus to unknown receptors tion or penetration. These can be considered 'pri-is mediated by the viral fibre protein. The virus then mary receptors'. In the absence of these primary uses Arg-Gly-Asp repeats in the penton base proreceptors viruses may use other cell surface com-tein to bind to vitronectin receptors (a b and a b ). v 3 v 5 ponents for entry (Fig. 1) . These components may be Conformational changes in the vitronectin receptor less efficient than the primary receptor. Nevertheless, are required for ligand-induced receptor internalisathey are competent for entry. Such molecules can be tion [157, 158] . It is unclear whether the penton base regarded as 'alternative receptors'. For SFV, MHC protein induces similar changes or triggers endoclass I antigens have been proposed as receptors cytosis by an alternative mechanism [243] . Thus [86] , yet this virus clearly infects MHC class I although the virus is able to adhere to the cell negative cells [154] , suggesting that at least one surface, a second receptor is essential for endocytosis alternative receptor exists. For the related Sindbis and infection. In cases such as these, we suggest that virus the laminin receptor has been implicated in the term 'co-receptor' is used when there is evidence virus attachment and entry [228] but it is unclear for direct interaction with the virions, and 'co-factor' whether this molecule also binds SFV. when the role of the second component is indirect or For most primate lentiviruses, CD4 and a unknown. chemokine receptor are required for entry. CD4 is sufficient to allow virus binding to cells, but a 3.4.1. Co-receptors for the primate lentiviruses chemokine receptor is required for fusion and in-Soon after the identification of CD4 as a receptor fection and can be regarded as a co-receptor for entry for HIV-1 it became apparent that additional cell (see below). Some strains of HIV, however, are able surface components were required for fusion and to infect CD4 negative cells. These viruses can use infection. Expression of human CD4 in murine NIH CXCR4, CCR3 or an orphan chemokine receptor 3T3 cells, for example, failed to elicit infection, V28 for entry [62, 174] . In these situations the whereas expression in HeLa cells rendered these chemokine receptors could be regarded as alternative cells permissive. Together the data suggested that receptors (provided they alone mediate binding and HIV-1 required at least one other factor for infection fusion). HIV-1 can also use galactosylceramide to [10, 34, 56, 119] . It was also recognised that there infect some neural and intestinal CD4 negative cell might be multiple accessory factors as the primate lines [17, 82] . Whether this glycolipid mediates entry lentiviruses were shown to differ in their capacity to 1ve itself or requires additional factors is unclear. How-infect different CD4 cells. HIV-1 isolated early in ever, it is regarded as an alternative, albeit ineffi-the course of infection can usually infect macrocient, HIV receptor. phages and primary T cells, but seldom T-cell lines. In contrast, many T-cell line-adapted strains of virus, 3.4. Co-receptors or viruses isolated from patients with advanced disease, do not infect macrophages [143] . For some viruses it is apparent that an initial The accessory factors were identified during 1996 receptor interaction has a tethering role and that and found to be members of a family of sevenadditional cell surface molecules must then be transmembrane, G-protein-coupled, chemokine rerecruited for subsequent steps in entry (Figs. 1 and ceptors (see Ref. [234] ). The initial suggestion that (C9) In contrast to (C) , some HIV Env proteins [62] have a capacity to interact with the co-receptor (blue) alone without the requirement of the primary receptor. Binding to the primary receptor can still occur but is not obligatory. chemokine receptors might be involved in virus entry chemokine stromal cell derived factor 1[SDF-1] came from the identification of CC (b) chemokines [19, 153] . Other chemokine receptors were later (RANTES, MIP-1a, and MIP-1b) as HIV-suppres-identified as co-receptors for various strains of HIV-1ve sive factors secreted by CD8 T cells [40] . Fur-1, HIV-2 and SIV. In particular, CCR5, which binds thermore, lymphocytes from individuals who had RANTES, MIP-1a, and MIP-1b, is used by primary been multiply exposed to HIV-1 but remained unin-macrophage tropic strains of HIV-1 and by SIV fected showed a resistance to infection by macro-viruses [33, 35, 54, 57, 120] , while CCR3 and CCR2b phage tropic forms of HIV-1 that was associated with can be used by dualtropic virus (virus which can increased secretion of RANTES, MIP-1a, and MIP-infect both lymphocytes and macrophages). For a 1b [164] . Subsequently, the chemokine receptor now discussion of chemokines and chemokine receptors called CXCR4 (previously termed LESTR, see Refs. [170, 172] . HUMSTR and Fusin) was identified as an entry The viruses that can be isolated early in infection, co-factor for T cell line-adapted HIV-1 [16, 65] . and that appear to be responsible for transmission, CXCR4 has since been shown to bind the CXC (a) are macrophage tropic and utilise CCR5 as a co-receptor [35, 52, 57, 179, 180] . A mutant allele of 4. Post-binding events CCR5 has been identified. This mutation causes a frameshift that truncates CCR5 after the fourth The adsorption of virus to the cell surface and transmembrane domain and the mutant protein is not interaction with specific receptors initiates the events expressed on the cell surface. Thus CD4-positive that lead to fusion and penetration. For viruses that cells from homozygotes for the CCR5 mutation are undergo fusion at the cell surface, receptor engagegenerally not susceptible to infection by macrophage ment may initiate the molecular rearrangements tropic viruses [113, 182] (though they can be infected leading to penetration. For viruses that fuse intracelby T cell line-adapted viruses). Together, these lularly, binding should result in uptake of virions findings indicate that CCR5 is important for HIV-1 into endocytic vesicles. transmission, and that infection of a population of CCR5 expressing cells is usually required for the 4.1. Penetration at the cell surface virus to establish infection. The precise mode through which these molecules The plasma membrane is the principal barrier a function together with CD4 to facilitate virus entry is virus must cross to access the cell. However, this unclear. As discussed above, the primate lentiviruses membrane may not be the only obstacle that the are all pH-independent for entry [129, 130, 201] . The virus has to tackle. Many cells contain a highly organisation of the fusogenic envelope glycoprotein developed cortical cytoskeleton below the plasma (Env) of these viruses resembles that of influenza membrane (see Ref. [123] ). This complex of actin HA (see below). During fusion Env (which contains filaments and actin binding proteins can be up to a membrane bound subunit-gp41 or TM, and a 100 nm in depth and has the capacity to exclude non-covalently associated soluble subunit-gp120 or ribosomes and other organelles from the area imme-SU) must undergo a conformational change which at diately adjacent to the plasma membrane. Eukaryotic least functionally resembles the acid-induced con-ribosomes have sedimentation coefficients of 80S 6 formational changes that trigger HA mediated fusion. and are approximately 4.2 3 10 kD in size; by However, the conformational changes in Env occur comparison the pre-integration complex of HIV has a at neutral pH. CD4 binding can induce conforma-sedimentation coefficient of about 160-300S. Thus tional changes in HIV-1 Env that lead to exposure of the cortical cytoskeleton has the potential to restrict epitopes associated with gp41 and the V3 loop of the movement of incoming viral capsids to regions gp120 [61, 183] , and may modulate the interaction deeper in the cell. SFV that has fused at the surface between gp120 and gp41 [141] . How these changes of baby hamster kidney cells (after a transient drop in relate to the fusion is still under investigation. the pH of the medium to activate fusion) will infect However, a view is emerging that CD4-induced these cells [85, 123] . However, when the same maconformational changes in Env, including reorienta-nipulation is performed on Chinese hamster ovary tion of the V3 loop, allow the Env / CD4 complex to cells the virus does not infect, though it can do so bind to a given chemokine receptor [215, 252] and through the endocytic pathway [123] . The cortical that the chemokine receptor can then complete the structures are developed to different extents in conformational changes in Env required for fusion different cell types. For example, in BHK cells the (Fig. 2) . In many respects it is likely that the cortex is not well developed but in CHO cells it can chemokine receptors are essential for virus entry, as be seen in electron micrographs. some tissue culture strains of virus can dispense with If the cortex is a barrier to penetration, viruses will the need for CD4 but still require an appropriate have developed strategies to by-pass this structure. chemokine receptor [62] . Thus the role of CD4 is to The endocytic route may provide one such mecharecruit the virus to the cell surface and induce the nism. Though the molecular basis is not established, formation of the chemokine receptor binding site on endocytic vesicles can pass through the cortex. In gp120 / SU. While it is now well established that Env, general, the endocytic pathway is a hostile environ-CD4 and a chemokine receptor are generally essen-ment for a virus, lysosomes being a primary site of tial in HIV entry, a specific role for additional cell-cellular degradation. The need to penetrate the cortex surface molecules cannot yet be ruled out. may be one of the driving forces that has led viruses to use low pH as a means to delay triggering fusion The size of particles that can be internalised until after endocytosis. Thus even for pH-indepen-through coated vesicles is likely to be limited. SFV dent viruses, endocytosed virions may be at an virions which are 65 nm in diameter are easily advantage, as far as traversing the cortex is accommodated in coated vesicles. The larger virions concerned, over virions that fuse at the cell surface. of influenza virus and HIV (approx. 100 nm diam-How do viruses that fuse at the cell surface eter) also enter these vesicles, as do those of rhabpenetrate the cortex? At present there is little in-doviruses such as VSV. However, these latter elliptiformation. In HIV, the virion-associated protease is cal virions, for which the long axis is approximately known to cleave cellular proteins in addition to its 150 nm, tend to be internalised slowly compared to own Gag gene products. Among these cellular SFV, possibly reflecting some difficulty in entering proteins are actin and several actin-binding proteins coated vesicles [127] . [1, [185] [186] [187] 213] . Perhaps lentiviruses use their pro-Non-clathrin mediated endocytosis has also been tease to penetrate the cortex. implicated in constitutive endocytosis [219] . The molecular mechanisms involved in the formation of 4.2. Endocytosis these vesicles are not well characterised. In some situations this pathway appears to be upregulated in Most cells have the capacity for endocytosis response to inhibition of clathrin-mediated endothrough several distinct mechanisms. Although endo-cytosis [45, 48] . It is unclear what the basal level and cytic routes for virus entry have long been proposed, function of the pathway is in non-perturbed cells. In it only became clear that endocytosis could be BHK at least, clathrin-coated vesicles mediate the essential for virus infection when the pathway of bulk of constitutive endocytosis [124] . SFV entry was determined [84] . Many cells can take up particulate ligands by Clathrin-coated vesicles are about 100 nm in di-phagocytosis [15] . Although most prominent in ameter and form by invagination of coated pits-specialised cells, such as monocytes and neutrophils, plasma membrane domains coated with a complex of phagocytosis is not limited to these cells. The clathrin and AP2 adaptors. Depending on the cell process is receptor-mediated and is initiated when type, many hundreds of these vesicles may form appropriate cell surface receptors are complexed by each minute through most of the cell cycle and are polyvalent ligands. Receptor ligation leads to the the main conduit for receptor-mediated endocytosis, activation of a signalling cascade involving tyrosine fluid-phase endocytosis and constitutive membrane kinases and other enzymes that drive the polymeriturn over [229] . Following budding, the clathrin coat sation of actin on the cytosolic face of the membrane is removed through the action of an uncoating adjacent to the adsorbed particle. This actin poly-ATPase and the vesicles fuse with early endosomes. merization pushes membrane around the particle, From endosomes, membrane and content can be allowing the engagement of more receptors and recycled to the cell surface or delivered to other culminating in the engulfment of the particle. Phagocellular destinations, including late endosomes and cytosis is strictly ATP dependent and can be blocked lysosomes, the Golgi apparatus and, in specialised by inhibitors of tyrosine kinase activity and actin cells, synaptic vesicles, MHC class II-containing polymerisation. compartments and alternative plasma membrane In general, phagocytosis is involved in the uptake domains (see Ref. [80] for review). Early endosomes of large particles, such as cells and opsonised are acidified to approximately pH 6, through the bacteria, that cannot be included within clathrinmembrane-associated vATPase, and late endosomes coated vesicles. Whether it has a role in the entry of and lysosomes can be more acidic. Internalised viruses is unclear. Influenza virions have been seen ligands en route to lysosomes will transit increasing-in uncoated vesicles with closely opposed vesicle ly acidic compartments. Viruses may exploit this pH membranes reminiscent of phagocytic vesicles [163] , gradient to facilitate their delivery to specific sites in and it is possible that larger virions such as the the cell [102] . poxviruses are internalised through phagocytic vesi-cles. Following phagosome formation these vesicles microtubule-organising centre and may interact with fuse with endosomes and lysosomes to acquire the ER [190] . vATPase and enzymes capable of hydrolysing the Morphological studies indicated that SV40 is phagocytic content. The acquisition of the vATPase taken up in small vesicles and delivered directly to again causes the phagocytic vesicles to become the ER / nuclear membrane [94] . Recent studies have acidic, thereby enabling the fusion potential of acid-shown that these vesicles are caveolae [5, 196] . SV40 dependent viruses to be activated. is believed to use MHC class I antigens as receptors [11, 25] . Early morphological studies showed that class I antigens could be associated with small non-4.2.3. Macropinocytosis clathrin coated invaginations of the plasma mem-Macropinocytosis involves the formation of, usubrane [91] . In retrospect, these invaginations might ally, large vesicles in regions of plasma membrane have been caveolae. However, class I antigens have ruffling [209] . The processes can be induced by also been implicated as receptors for SFV [86] , a treating cells with growth factors or phorbol esters, virus that clearly uses the clathrin-mediated route for but appears to be constitutively active in macroendocytosis [124] . Thus MHC class I antigens might phages and dendritic cells, where it has been impliinternalise through different endocytic routes [118] , cated in a pathway for presentation of exogenous or different viruses might direct these receptors to antigens on MHC class I molecules [150, 151] . As specific endocytic carriers, possibly through associawith phagocytosis the formation of macropinocytic tion with other co-receptor molecules. Although vesicles involves actin polymerization, and can lead implicated in SV40 entry, a role for caveolae in the to delivery of internalised ligands to late endosomes uptake of other viruses remains to be established. and lysosomes. However, the process is not liganddriven nor does it appear to require activation of 4.3. Fusion mechanisms specific receptors. The role of macropinocytosis in viral infection is unknown. However, constitutive Regardless of the location, the critical event in macropinocytic activity may predispose monocytes penetration for enveloped viruses is fusion. Currentand dendritic cells to infection by viruses such as ly, the fusion reactions triggered by influenza HA in HIV [30, 106] . particular but also the spike glycoprotein of SFV have been dissected in considerable detail. The 4.2.4. Caveolae influenza virus system provides the best model for Caveolae are small 50 nm flask-shaped plasma cellular membrane fusion (for a more extensive membrane invaginations that have long been recog-discussion of viral fusion mechanisms see Ref. nised in capillary endothelia and other cells. Recent [14, 21] ). evidence suggests these structures are more widespread and that they have a role in the clustering and 4.3.1. Influenza virus fusion signalling pathways associated with GPI-linked and other membrane proteins [112] . The integrity of 4.3.1.1. Structure of the influenza virus particle. caveolae appears to depend on the presence of The influenza virus envelope carries three different cholesterol and a cholesterol-binding protein Vip21 / transmembrane proteins (i) HA (approx. 500 copies caveolin [161] . Initially, it was proposed that these per virion), (ii) the neuraminidase (NA-approx. 100 structures might transiently invaginate, in a process copies per virion), and (iii) the M2 protein (a small termed photocytosis [6]. More recently it has become tetrameric protein present in low copy number per apparent that caveolae can undergo actin-dependent virion) [90, 206] . Of these, HA alone is required for internalisation in cells treated with the phosphatase fusion. inhibitor okadaic acid [162] . The role of this internalisation is uncertain and there is no evidence that 4.3.1.2. Haemagglutinin (HA) synthesis and struccaveolae are delivered to the endocytic pathway. ture. However, internalised caveolae appear to be relo-HA is assembled in the ER of infected cells from a cated to the perinuclear region adjacent to the single precursor polypeptide (HAO) of approximate-ly 560 amino acids. The protein undergoes N-linked membrane fusion. The protein can be purified to glycosylation, trimerisation and folding within the homogeneity from virus suspensions and is fusion-ER, and only when the protein has folded correctly is competent when reconstituted into artificial memit released by the ER quality control system for branes or when expressed alone in cells in the transport to the Golgi and plasma membrane. The absence of other viral proteins. Furthermore, a water folding and assembly of HA have been studied soluble bromelain-cleaved fragment of HA (BHA; extensively and involve the combined activities of a the fragment initially used to derive the crystal set of ER chaperones [24, 122, 210] . En route to the structure of the neutral pH conformation), though not cell surface the oligosaccharides are modified to fusion-competent, will undergo a number of the complex forms, and each HAO molecule is conformational changes associated with fusion. proteolytically cleaved by furin or furin-type en-Studies directly correlating the kinetics of the zymes in the trans Golgi cisternae or trans Golgi pH-induced changes in the conformation of HA with network to generate the HA1 and HA2 subunits. The membrane fusion suggest the following sequence of X-ray structure of HA shows that the mature protein events. Exposure to low pH induces a rapid change contains two domains (i) the globular heads assem-in the tertiary structure of HA involving at least two bled from HA1 containing the sialic acid-binding steps. The first is the exposure of the HA2 fusion sites and (ii) a stalk domain composed primarily of peptide which may insert into the target membrane HA2 which supports the head domains. After cleav- [198] (and possibly in the viral membrane as well age, the newly generated N terminus of HA2 con- [230, 237] ). After a short lag phase, lipid mixing can tains a stretch of conserved hydrophobic residues be detected. Finally, the HA1 subunits dissociate, as that is termed the 'fusion peptide'. This segment is detected by (i) the loss of epitopes formed by crucial for fusion; non-cleaved HA molecules are not adjacent HA1 subunits, (ii) the exposure of epitopes fusion-competent. In the neutral pH form of HA, the initially facing the inside of the stem, and (iii) the fusion peptide is buried away from the solvent within unmasking of proteolytic sites in HA1 the stalk structure about 3.5 nm from the viral [76, 96, 199, 241] . membrane. The cleavage in part provides a mecha-How exactly the changes in HA occur and how nism to allow the HA trimer to transit the acidic they allow the molecule to interact with membranes compartments of the exocytic pathway, but it is also to induce fusion is still unclear. The position of the likely that correct folding and assembly of trimers fusion peptide at neutral pH close to the viral can only be achieved with the full length precursor membrane initially posed a topological problem: protein. The HA2 sequences form two long anti How could the fusion peptide interact with the target parallel a helices linked by a loop. This region and membrane, when it is located 100 A away from it? its flanking a helices contain several leucine-zipper Modelling studies suggested that during fusion, when heptad repeats. In other proteins similar sequences the loop structures of adjacent HA2 molecules are show a propensity to form coiled coils. As discussed released by the protonation of residues involved in below it appears that at least part of the HA maintaining the trimeric structure, they would refold conformational change during fusion involves the to form an energetically more favourable triple refolding of this loop region to form a coiled coil. stranded coiled coil. This change would create a long Thus the HA protein can be regarded as being extended helix and thereby relocate the fusion pep-'spring-loaded', i.e. the full length HAO is assem-tide 100 A from within the stalk to the end of the rod bled in a metastable conformation that can refold, where it could potentially interact with a target after cleavage, when a critical stimulus (exposure to membrane [31] . Support for this model came with low pH) is applied. the resolution of the 3D structure of the acid form of a fragment of HA2 [29] . This structure showed 4.3.1.3. HA-mediated membrane fusion. reorganisation of the loop to an a-helix and reloca-HA thus forms 13.5 nm rod-like structures that are tion of the fusion peptide to the tip of the molecule. expressed on the cell surface and incorporated into However, additional changes occur at the juxtavirions during assembly. Many features have made membrane end of the molecule. Part of the long helix this protein an ideal tool to study protein-induced in the neutral-pH structure is relocated such that it becomes anti-parallel to the rest of the helix. Thus pressure on the bilayer to drive a hemi-fusion the longitudinal helix of HA2 is prolonged and intermediate to full fusion [131] . Full fusion straightened out at the N terminus and truncated by proceeds through the formation of a narrow (1-2 nm bending at the C terminus [29] . Evidence supporting diameter) pore that can be detected by the patch these changes in native HA has been gained from clamp technique [194, 195, 216] . The initial pore electron microscopy [237] . However, information on formation follows after a short lag, but precedes lipid the structure of the C terminal region of HA2 is still mixing [216] . The initial fusion pore flickers open missing and the orientation of the acid form of the and closed [194, 195] before widening further to protein with respect to the viral membrane is uncer-allow first the movement of lipids and subsequently tain. The EM studies, like earlier photo-affinity the aqueous contents of the fusing cells to mix [20] . labelling studies [230] , suggested that the fusion peptide might interact with the HA-containing mem-4.3.2. Semliki forest virus brane, i.e. that at some stage at least the acid form of the protein might be inverted. 4.3.2.1. Structure and synthesis. Together the data suggest that exposure to low pH Limited X-ray crystallographic data exist for the might allow the fusion peptides to swing out from alphaviruses. Nevertheless, structural information has their locations within the HA2 stalk, perhaps driven been gained from image analysis of cryo-preserved by the initial events involved in refolding the loop viruses by EM [72, 220] . Alphaviruses, such as SFV region. These events may culminate in the form of and the closely related Sindbis virus, are uniform in the protein seen in the low-pH crystal structure, but size (approximately 65 nm diameter) and regularly the fusion-competent form could be an intermediate shaped. The SFV nucleocapsid comprises one RNA in this series of changes. If, after exposure of the molecule and 240 copies of the capsid (C) protein. fusion peptide, the HA protein is tipped, the fusion The capsid proteins interact with the viral RNA and peptides could interact with both the target mem-each other to form an icosahedral nucleocapsid with brane and the virion membrane, in keeping with a T 5 4 surface lattice [36, 159, 160] . The SFV enlabelling studies [197, 231] , i.e. the protein may adopt velope contains 80 spike glycoproteins, each spike a tilted conformation parallel to both the viral and consisting of three E2E1 heterodimers [a small the target membrane [81, 197, 200, 251] . Electron peptide, E3, resulting from proteolytic cleavage of paramagnetic resonance studies have suggested that P62 (see below) is lost from Sindbis virus spikes but some or all of the coiled coil could insert into remains non-covalently attached to the SFV spike membranes [257] . However, photo affinity labelling complex]. The spikes are triangular and form, like indicates that only the fusion peptide is inserted [59] . the nucleocapsid, an icosahedral shell with a T 5 4 How changes in HA induce the reorganisation of surface lattice [71, 220] , suggesting that each spike the opposing lipid bilayers is also still unclear. It is glycoprotein heterodimer directly interacts with a unlikely that fusion pores are formed from a single capsid protein [132, 207] . This interaction appears to HA trimer [38, 60] . Current data suggest that a be mediated through a conserved tyrosine in the minimum of three HA trimers is required to form a cytoplasmic domain of E2 and a hydrophobic pocket functional fusion complex [49] and that the trans-on the capsid protein [188, 189] . Chemical crossmembrane domain of HA is crucial for fusion. Using linking studies on Sindbis virus suggest that each cell-to cell fusion assays, an HA molecule anchored spike contains an inner core of the three E1 subunits to the membrane by a GPI moiety, rather than its and an outer shell formed by the three E2 subunits, usual transmembrane domain, will induce hemi-fu-an arrangement confirmed by cryo-electron microsion-a state in which the outer but not the inner scopy [8, 72] . A receptor-binding site in the spike leaflets of the bilayers are fused [97] . Experiments complex has not yet been identified. However, a with amphipathes that selectively insert into either hydrophobic domain in E1 between amino acids 79 the inner or outer leaflet of membranes and change and 97 (for SFV) is highly conserved in alphaviruses the curvature of that membrane, suggest that the [98] , and is believed to be involved in fusion normal transmembrane domain is required to exert [58, 110] . As with HA, alphavirus spike proteins are assem-activation indicate that a cavity in the neutral pH bled in the ER, and transported to the cell surface spike complex is narrowed by the centripedal movewhen folding is completed. These spike proteins are ment of the E1 subunit which is extended towards a also synthesised in precursor form. The E1 protein target membrane. In contrast, the E2 subunits sepaassembles together with the precursor of E2, the P62 rate [72] . A role for these changes is supported by protein. Three such dimers subsequently assemble to the ability of a monoclonal antibody that recognises form a spike protein complex that is transported to an epitope exposed on the low pH form of E1 to the cell surface. Just prior to its arrival at the plasma inhibit fusion and infection [72, 223, 224] . membrane, the P62 protein is cleaved by a cellular The structural changes precede the onset of fusion, protease to generate the E2 protein found in the as monitored by lipid mixing [28] . Following the mature spike glycoprotein [50] . The properties of the conformational changes described above, the virus mature E2E1 heterodimers are distinctly different acquires the capacity to bind to cholesterol-confrom the precursor P62E1 dimers. Exposure of E2E1 taining lipid vesicles [99, 149, 167, 238] . Though SFV complexes to mildly acidic conditions results in will bind to cholesterol-containing liposomes at low dissociation of the subunits, whereas P62E1 com-pH, it will not fuse with these membranes unless plexes dissociate only upon exposure to pH values of they contain low amounts (about 1 mol%) of sphing-4.5-5.0 [114, 115, 181, 222] . Thus P62 appears to olipids [43, 138, 149] . Ceramide is the minimal strucstabilise the immature spike complex and prevent tural component of the sphingolipids that support premature activation during transport through the fusion [149] . SFV E1 interacts with both cholesterol mildly acidic compartments of the exocytic pathway. [99] and sphingolipids [138] in a stereo-specific The cleavage of P62 may also induce a conforma-manner. How these lipids promote SFV fusion is not tional change in the heterodimers, resulting in the known, but they may promote additional conformaformation or exposure of receptor-binding sites: SFV tional changes that are required for fusion or prevent particles, carrying spikes with an uncleaved P62, the inactivation of the fusion-active form of E1. have a reduced capacity to bind to cells [180] . Thus, proteolytic cleavage of the SFV spike glycoprotein 4.3.3. Other viruses controls both the activation of the viral fusion A pattern that emerges from studies on SFV and potential and the viral receptor-binding capacity. The influenza virus is that conformational changes ininteraction of Sindbis virus with its cell surface duced by low pH lead to the formation of stable receptor may initiate additional conformational trimeric complexes of subunits of the envelope changes in the spike glycoprotein [67, 133, 134] . proteins (E1 and HA2, respectively). Studies of the Although these changes might have some role in Flavi virus tick-borne encephalitis virus (TBE), for enhancing the binding, or in preparing the spike which a 3D structure of the VAP/ fusion protein has protein for fusion, they are unlikely to be essential been resolved [175] , suggest that this molecule also for fusion: SFV, at least, will fuse with pure lipid rearranges to form stable trimers during low pHmembranes when the pH is lowered appropriately induced fusion [3, 175] . In contrast to SFV and [238] . influenza virus, in which the spike protein and HA respectively form trimeric structures that project out 4.3.2.2. SFV fusion. from the membrane, the TBE envelope protein is a Following exposure to low pH the rearrangements dimer that is oriented parallel to the viral membrane. in the SFV spike protein are initiated by the dissocia-Thus for this virus, trimer formation during fusion tion of the E2 subunits from E1 [115, 180, 224] . The involves extensive reorganisation of the fusion proreleased E1 monomers then reorganise into homot-tein subunits. rimers [28, 223, 224] . The masking of trypsin cleavage sites on E1, and changes in the antigenicity of 4.3.3.1. Retrovirus fusion. epitopes on E1 [28, 100, 101, 180, 224] parallel the In most cases the retroviruses are pH-independent reorganisation of the spike protein observed mor-for fusion. As discussed above for HIV, it is likely phologically. Time-resolved images of SFV after pH that some aspect of the envelope protein-receptor interaction activates fusion. Binding of the ALV-A and current comparisons to HA should be regarded receptor to the viral envelope protein complex has with some caution. Nevertheless, several studies been shown to induce conformational changes in the have suggested that the retroviral envelope proteins SU-TM complex [75] . Several of the primary re-form trimers [18, 64, 232] . The retroviral transmemceptors for retroviruses, as well as the co-receptors brane proteins contain N terminal hydrophobic sefor the primate lentiviruses, are multi-membrane quences that resemble the HA fusion peptide (Fig. spanning proteins, suggesting that some property of 3). Furthermore, domains with heptad repeats have this class of protein might facilitate retroviral fusion. been implicated in fusion [244, 247] . Synthetic pep-However, at least two retroviruses (bovine leukaemia tides representing sequences C-terminal to the fusion virus and avian leukosis virus) do not use multi-peptide have a strong propensity to form a helices in membrane spanning protein receptors (Table 1) . solution [103, 246] . Hence, it has been speculated Similarities in the organisation of retroviral en-that receptor binding can unleash conformational velope proteins and influenza HA suggest that as-changes that involve the formation of coiled coils pects of retrovirus and orthomyxovirus fusion may and exposure of the TM fusion peptide be similar. However, the 3D structure of a native [18, 64, 116, 245] . Synthetic peptides representing the retroviral envelope protein has not yet been solved HIV heptad repeats can block virus entry, perhaps by The two precursor polypeptide chains are represented from their N-termini to the left to their C-termini to the right. The HIV Env encompasses approximately 860 residues and influenza HA 560. The C-termini of both molecules are cytoplasmic, or intravirional, but the cytoplasmic domain of HIV Env is considerably longer than that of HA (as represented by the segments to the right of the trans-membrane domains). During transport to the cell surface both precursors are cleaved as a prerequisite for subsequent fusion activity. The two resulting polypeptide chains of HIV Env, SU / gp120 (surface) and TM / gp41 (transmembrane), are non-covalently associated. Those of influenza virus, HA1 and HA2, are linked by a disulphide bond (intrachain disulphide bonds are not marked). SU and HA1 are responsible for the binding of the respective viruses to cell surface receptors, CD4 and sialic acid. Both receptor-binding sites are created by the juxtaposition in space of conserved residues that are non-contiguous in the amino-acid sequence. In SU of HIV these residues are situated in conserved regions on both sides of the V3 loop. The V3 loop influences the interactions with the chemokine-receptor co-receptors. The cleavage of the precursors creates novel N-termini in the transmembrane proteins. These N-terminal peptides share similar hydrophobic sequences and have been implicated in membrane fusion. C-terminal to the fusion peptides are potential 'coil regions', two in the HIV TM extracellular domain (approx. residues 550-580 and 630-660 in the precursor), and one region in HA2 (residues 40-105). These regions have a propensity to form alpha helices and may form coiled-coils as a step in a series of events that activate the membrane-fusing capacities of the two proteins. interfering with the ability of these regions to influenza virions to low pH, proton influx through oligomerise during the fusion reaction [128] . The M2 acidifies the interior of the virion. Acidification structure of an alpha helical domain from the ex-of the viral core is presumed to disrupt the pHtracellular portion of HIV-1 TM has been solved sensitive interaction of matrix protein (M1) and the crystallographically [32] . This domain forms trimers RNA binding nucleoprotein (NP) [126, 259, 260] , of two interacting peptides. The N-terminal peptide, thereby allowing the viral RNPs to be transported to which contains heptad repeats, forms a trimeric the nucleus [83, 125, 126] . Amantadine, a weak base coiled coil. Around this coiled coil the C-terminal that accumulates in endosomes, interrupts this sepeptides are wound such that they fit into the quence of events by blocking the channel function of hydrophobic grooves on the coiled coil. This struc-M2 [77, 83, 126, 168, 202, 205, 206, 226] . ture might position the HIV fusion peptide at the top of a protruding six helix bundle such that the fusion 5.2. SFV peptides could insert into a membrane. The structure shares some features with that of the low pH The intracellular part of the alphavirus replication fragment of influenza virus HA and is suggested to cycle takes place entirely in the cytoplasm of infecrepresent the core of the fusion active gp41. that replication occurs in association with membranes of the late endosomes, lysosomes and ER [69] . Thus the viral RNA appears to remain associ-5. Early post-penetration events ated with the same compartments from which the virus fuses. After fusion the fate of the nucleocapsid varies according to the destination of the virus within the 5.3. HIV cell. Multiple mechanisms, illustrated by the systems discussed below, demonstrate the ability of different In contrast to most retrovirus, HIV will infect viruses to exploit the properties of host cells for their non-dividing cells [203] . This indicates that the HIV own special requirements. For example, the DNA proviral DNA, which has been reverse-transcribed genome of HSV must be delivered to the nucleus. from the RNA genome, is able to penetrate the intact Although many of the molecular details remain to be nuclear membrane. The HIV pre-integration complex established, it seems that following fusion some of contains the viral matrix (MA) protein [203] . Recent the tegument proteins are removed but the capsid studies have identified nuclear targeting signals in remains intact, binds cytoplasmic dynein and uses MA similar to those previously characterised in other the cellular microtubule system to translocate itself cellular and viral proteins. MA is linked to the to the perinuclear region. In this location the capsids plasma membrane via an N-terminal myristic acid bind directly to the nuclear pore and the DNA is group. However, MA can be phosphorylated on delivered to the nucleus without complete disassemb-multiple serine and tyrosine residues. Phosphorylaly of the capsid [193] . tion displaces MA from membranes, in a manner similar to that demonstrated for the MARCKS 5.1. Influenza virus protein [212] . Thus MA-containing viral pre-integration complexes are likely to be released from the In the case of influenza virus acidification of the plasma membrane by phosphorylation of MA. The viral core is required for cellular infection. The nature of the kinase(s) involved in these phosphorylenvelopes of influenza virions contain small numbers ation events is unclear, but serine / threonine and of M2 protein molecules that function as proton tyrosine kinase activity has been detected in highly channels [26, 37, 126, 205, 257, 258] . On exposure of purified HIV virions [203] . Thus the virus may package cellular kinases during assembly to facilitate proteins have proven to be effective delivery vehicles its subsequent targeting to the nucleus. for DNA, drugs, peptides and toxins in tissue culture. How the released complex is transported to the Initial studies suggest some of these also work in nuclear membrane where the nucleophilic signals animal models for the delivery of peptides to the presumably allow interaction with the nuclear pore is cytoplasm of cells and presentation on MHC class I unclear. As discussed above the cortical cytoskeleton antigens. Future studies will undoubtedly allow more may present a barrier that the viral capsid has to effective and more useful vectors to be developed. traverse (Section 4.1). Other virally encoded proteins including integrase and the product of the vpr gene are also present in the pre-integration complex, though the capsid protein is not. Vpr also appears to Acknowledgements be required for infection of non-dividing cells. Nuclear targeting signals have not been identified in The authors are supported by grants from the this protein, although it is karyophilic [73] . It is not United Kingdom Medical Research council. impossible that Vpr and / or other proteins in the pre-integration complex function in bringing the incoming viral pre-integration complex from the References plasma membrane to the nucleus through interaction with cellular transport pathways [203] . reached the stage of clinical trials (for reviews see Virol. 69 (1995) 695-700. Refs. [7, 135] ). Vectors based on retroviruses, adeno- [4] K.B. Andersen, H. Skov, Retrovirus-induced cell fusion is enhanced by protease treatment, J. Gen. Virol. 70 (1989) and herpesviruses, which will allow stable integra- 1921-1927. tion and expression of a given gene, have proved I-expressing cells [121, 218] . Virosomes containing of nonprimate cells with human cell, J. Virol. 64 (1990) reconstituted influenza, Sendai, VSV or EBV fusion 2149-2156. 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